AP Biology

AP Biology

Lecture #36Bacterial Transformation

DNA Technology & Genomics

A Brave New World

TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTCCTAAAGATATCCGGAATAT

human genome3.2 billion bases

• Genetic Engineering– manipulation of DNA– if you are going to engineer DNA & genes &

organisms, then you need a set of tools to work with

– this unit is a survey of those tools…

Biotechnology today

Our tool kit…

• Bacteria review – one-celled prokaryotes– reproduce by mitosis• binary fission

– rapid growth• generation every ~20 minutes• 108 (100 million) colony overnight!

– dominant form of life on Earth– incredibly diverse

Bacteria

• Single circular chromosome– haploid– naked DNA• no histone proteins

– ~4 million base pairs• ~4300 genes• 1/1000 DNA in eukaryote

Bacterial genome

How have theselittle guys gotten to be so diverse??

• Small supplemental circles of DNA• 5000 - 20,000 base pairs• self-replicating

– carry extra genes• 2-30 genes • genes for antibiotic resistance

– can be exchanged between bacteria• bacterial sex!!• rapid evolution

– can be imported from environment

Plasmids

• A way to get genes into bacteria easily– insert new gene into plasmid– insert plasmid into bacteria = vector– bacteria now expresses new gene• bacteria make new protein

How can plasmids help us?

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transformedbacteriagene from

other organism

plasmid

cut DNA

recombinantplasmid

vectorglue DNA

• Plasmids used to insert new genes into bacteria

Biotechnology

gene we want

cut DNA

cut plasmid DNA

insert “gene we want” into plasmid...“glue” together

ligase

like what?…insulin…HGH…lactase

Cut DNA?DNA scissors?

recombinant plasmid

• Restriction enzymes– restriction endonucleases– discovered in 1960s– evolved in bacteria to cut up foreign DNA • “restrict” the action of the attacking organism• protection against viruses

& other bacteria– bacteria protect their own DNA by methylation & by not

using the base sequences recognized by the enzymes in their own DNA

How do we cut DNA?

What do you notice about these phrases?radarracecarMadam I’m AdamAble was I ere I saw Elbaa man, a plan, a canal,

PanamaWas it a bar or a bat I saw?go hang a salami I’m a lasagna

hog

palindromes

Restriction enzymes• Action of enzyme – cut DNA at specific sequences• restriction site

– symmetrical “palindrome”– produces protruding ends• sticky ends• will bind to any complementary DNA

• Many different enzymes– named after organism they are found in• EcoRI, HindIII, BamHI, SmaI

Madam I’m Adam

CTGAATTCCGGACTTAAGGC

CTG|AATTCCGGACTTAA|GGC

Discovery of restriction enzymes1960s | 1978

Werner Arber Daniel Nathans Hamilton O. Smith

Restriction enzymes are named for the organism they come from:EcoRI = 1st restriction enzyme found in E. coli

Restriction enzymes• Cut DNA at specific sites– leave “sticky ends”

GTAACG AATTCACGCTTCATTGCTTAA GTGCGAA

GTAACGAATTCACGCTTCATTGCTTAAGTGCGAA

restriction enzyme cut site

restriction enzyme cut site

Sticky ends• Cut other DNA with same enzymes– leave “sticky ends” on both– can glue DNA together at “sticky ends”

GTAACG AATTCACGCTTCATTGCTTAA GTGCGAA

gene you want

GGACCTG AATTCCGGATACCTGGACTTAA GGCCTAT

chromosome want to add gene to

GGACCTG AATTCACGCTTCCTGGACTTAA GTGCGAA

combinedDNA

Sticky ends help glue genes togetherTTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTTAACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA

gene you want cut sitescut sites

AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGC

TTACCAATGAACATTGCTTAAGATGCTAGCGGCTAAGTTGCGA

chromosome want to add gene tocut sites

AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene

sticky ends

chromosome with new gene addedTAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC ATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC

sticky ends stick togetherDNA ligase joins the strands

Recombinant DNA molecule

Why mix genes together?

GAATTCTACGAATGGTTACATCGCCGAATTCTACGAT CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTA

• Gene produces protein in different organism or different individual

aa aaaa aa aa aa aa aa aa aa

“new” protein from organism ex: human insulin from bacteria

human insulin gene in bacteria

bacteria human insulin

How can bacteria read human DNA?

The code is universal• Since all living

organisms… – use the same DNA– use the same code

book– read their genes the

same way

Copy (& Read) DNA• Transformation– insert recombinant plasmid

into bacteria– grow recombinant bacteria in agar cultures • bacteria make lots of copies of plasmid• “cloning” the plasmid

– production of many copies of inserted gene– production of “new” protein• transformed phenotype

DNA RNA protein trait

Grow bacteria…make more

growbacteria

harvest (purify)protein

transformedbacteria

plasmid

gene fromother organism

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recombinantplasmid

vector